Measurement of fluorescence intensity, spectrum, lifetime, and polarization can determine the presence of fluorophores and their concentrations. The equipment used is called fluorometer, it is an analytical instrument designed for the determination of the concentration of fluorescent compounds. The technique of analysis is called fluorometry, it is widely accepted in the scientific field, and is a valuable and powerful analytical tool, both for quantitative and qualitative analysis. It is a sensitive optical emission technique, consisting of exciting the sample molecules with a photon source. Fluorometry is still being explored, modern instruments offer multiple advantages that facilitate data collection. It is generally used if there is not a sufficiently sensitive or selective colorimetric method for the substance to be determined.
Benefits of using the fluorometer
- Sensitivity: most analytes allow a part detectability index per trillion, the detection limit depends largely on the sample being measured. This feature allows detection using small amounts of samples. Fluorometry, due to its high sensitivity, is used to quantify DNA, RNA and PCR, to detect DNA contamination in drugs and to perform vitamin tests for pharmaceutical procedures.
- Specificity: Fluorometers are highly specific because they are the target compounds that absorb and emit light, making them less susceptible to interference from other aggregates present in the sample. This instrument allows us to identify the presence and the amount of specific molecules in a medium.
- Linearity in the concentration range: The intensity of the fluorescence is usually directly proportional to the concentration of the absorber. The concentrations of unknown biomolecules can be mathematically quantified against the fluorescent intensity of the known biomolecules within the assay. Modern fluorometers can detect concentrations of fluorescent molecules as low as 1 ppt, the operating wavelength range of the fluorometer depends on the requirements of the experiment.
- Simplicity and speed of response: the sensitivity and specificity of the fluorometer allow to reduce experimental protocols for sample preparation that for other methods of analysis are rigorous. For this reason, fluorometry is a relatively simple analytical technique and allows the analysis to be simple, practical and have a rapid response.
- Economic: Costs associated with reagents and instrumentation are low, relative to other analysis techniques. Fluorometry is a powerful technique used for a variety of environmental, industrial, diagnostic, DNA sequencing, forensic, genetic and biotechnological applications. The fluorometer is a device that can be found in various presentations that allows them to meet the demand of the research itself.
Filter Fluorometers
The device has an excitation source, filters, sample cells and fluorescence detectors. Filter fluorometers have a single beam configuration, with source intensity control to reduce the effects of fluctuations in the detector response. These sources can produce intense radiation of 254 nm, the most common light source being xenon arc lamps. The selection of the excitation wavelength is performed by inserting a primary filter in the incident beam with this a spectrum with good spectral purity is obtained.
Filter fluorometer is a good choice when sensitive quantitative measurements for specific compounds are desired. This device provides a relative measurement and can be calibrated to a known concentration standard or correlated with other laboratory methods. Modern microprocessor-based instruments offer a variety of data manipulation possibilities through the software. They include data processing and display options, blank signal subtraction, derived spectra, spectrum filtering, peak detection, parameter statistics.
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